The goal of this proposal is to test if our recent discovery on lipid trafficking in epithelial cells can be translated to clinical applications. Specifically, we will test the utility of using "short or unsaturated" ceramide-based lipids as molecular carriers to deliver therapeutic peptides or vaccine adjuvants across mucosal epithelial barriers. Mucosal surfaces represent vast areas where host tissues are separated from the environment only by a delicate but highly effective single layer of columnar epithelial cells, joined by tight junctions that are impermeable to proteins and even small peptides. So far, the lack of rational and efficient methods to circumvent this barrier has prevented the application of most therapeutic proteins for oral or nasal drug delivery and for mucosal vaccines. In the course of our studies on the biology of the glycolipid receptor for cholera toxin, ganglioside GM1, we recently discovered that the structure of the ceramide (lipid) domain dictates GM1 trafficking in epithelial cells. When applied apically, GM1-ceramides containing "short" C12:0 or "kinked chain" unsaturated C16:1 fatty acids (GM1short/unsat) enter the common/recycling endosome. Here, they are sorted for transport to various intracellular destinations and into the "transcytotic" pathway to the basolateral cell surface. In contrast, the GM1-ceramides with fully saturated fatty acid chains (C16:0 or longer) (GM1 long/sat) are instead transported to the late endosome and lysosome for degradation. In this exploratory project, we will test whether this basic discovery can be harnessed for transepithelial delivery of a bioactive peptide or protein adjuvant, both of which have clinical applications. We will link GM1 molecules containing unsaturated "kinked" or saturated fatty acids to the therapeutic peptide hormone glucagon-like peptide-1 (GLP1), which acts to regulate blood sugar (Aim 1);and to the TLR5-agonist FliC (Salmonella flagellin), which we use here to model a mucosal vaccine adjuvant (Aim 2). These studies will test a novel platform for transport of therapeutic/vaccine molecules across mucosal surfaces. There is great need for non-parental delivery of therapeutic peptides and proteins. Improved mucosal vaccine strategies are greatly needed for protection against pathogens, the vast majority of which invade via mucosal surfaces. PUBLIC HEALTH RELEVANCE: The goal of this application is to develop a way to allow for oral or nasal administration of therapeutic proteins and vaccines. Normally such proteins are not absorbed and must be delivered by injection;severely limiting therapeutic applications.